La personalidad Grit y su relación con otras variables psicológicas

Glutathione Transferase Omega is an atypical GST with minimal functional resemblance with the other members of the superfamily [179, 197, 262]. The gene is located adjacent to GSTO2 on Chromosome 10q24.3 and spans 1.5kb in length. GSTO1-1 was identified as a GST a decade ago due to its close sequence and structural similarity with the family and its ability, though minimal, to catalyse classic detoxification reactions with common GST substrates. Over the years, several atypical functions have been discovered (listed in Table 1.2), which will be discussed in detail in the sections to follow. Immunohistochemical studies identified GSTO1-1 in a range of diverse tissues with varied expression levels [263]. Though a surprisingly high expression level was observed in the heart and liver, the functional implications of the tissue-specific distribution are not fully understood. The study also reported the interesting subcellular localization of GSTO1-1 in the nucleus and nuclear membrane in several tissues. The significance of this unusual nuclear translocation is yet to be investigated.

36 The GSTO1-1 dependent regulation of IL-1β resulting from either the direct post translational modification of IL-1β or interaction with the inflammasome ASC complex offers a potential target pathway to study the deglutathionylating activity of GSTO1-1 [264, 265]. More recently, Kim et al. identified ATP synthase β subunit as an in vivo

target of GSTO1-1 in a Drosophila model of Parkinson’s Disease (loss of function mutation in the parkin gene) [261]. The transgenic fly model constitutively down- regulates the expression of GSTO1-1 and glutathionylated form of ATP synthase β subunit. Over expression of GSTO1-1 in the parkin mutants was shown to increase glutathionylation of ATP Synthase and partially restore enzyme activity. Though the study suggests the catalytic role of Drosophila GSTO1-1 in glutathionylation, we propose that the deglutathionylating activity of GSTO1-1 may also be physiologically significant.

1.10.1 Atypical functions of GSTO1-1

GSTO1-1 was previously shown to catalyse the reduction of dehydroascorbate and exhibits low thioltransferase activity [262, 266]. These reactions are not catalysed by other GSTs but are characteristic of glutaredoxins. The functions unique to GSTO1-1 are listed in Table 1.2. Recent studies have demonstrated the significance of GSTO1-1 expression in cell defence mechanisms [260, 264, 267]. A study by Laliberte et al.

identified GSTO1-1 as a target of Cytokine Release Inhibitory Drugs (CRIDs) and hence implicating GSTO1-1 in mechanisms activating proinflammatory Interleukin 1β in monocytes [264]. The exact role of GSTO1-1 in this process is yet to be determined though a recent study in 2011 identified a novel interaction of GSTO1-1 with ASC (Apoptosis associated speck-like protein containing a CARD), a component of the inflammasome complex required for the activation of IL-1β production indicating the probability of an indirect effect of GSTO1-1 on IL-1β via protein-protein interactions within the activation complex [265].

There are several lines of evidence implicating GSTO1-1 in drug resistance in cell lines although no mechanism has been established [258, 259, 268]. Another study successfully linked GSTO1-1 over-expression with increased resistance to Cisplatin in HeLa cells [260]. On a different note, the redox sensitive regulation of cardiac Ryanodine receptor (RyR2) was reported by Dulhunty et al. [269]. Though the regulation of ryanodine receptors by GSTs is well established, the presence of cysteine at the active site of GSTO1-1 as opposed to Tyr/Ser increasingly questions the exact mechanism of RyR regulation by GSTO1-1. The redox sensitivity of GSTO1-1

37 dependent regulation of RyR would be an interesting option to explore with respect to determining the ‘mechanism of action’ of GSTO1-1.

Due to its structural similarity to glutaredoxin and its inability to recognize and strongly bind to typical GST substrates, we believe that its primary function is yet to be defined. Based on recent findings, my project proposes that GSTO1-1 may regulate a variety of proteins irrespective of their function via a common mechanism. The relevance of this hypothesis to my project is further elaborated in the following sections.

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Cell/Tissue/Animal model

Functional implications Reference

Monocytes Activation of IL-1β [264],[265] T47-D breast cancer cells,

rat hepatic cytosol

Reduction of

S- (phenacyl)glutathiones

[270, 271] HeLa Resistance to Cisplatin [260] Humans Delay in onset of neurodegenerative

diseases such as Parkinson’s and Alzheimer’s diseases

GSTO1-1 is implicated in Chronic obstructive pulmonary disease. Lower levels of GSTO1-1 reported in COPD patients when compared to non- smokers.

[214-216, 272] [213]

Calcium channels in lipid bilayers

Regulation of cardiac Ryanodine receptor RyR2

[269]

Caenorhabditis elegans Participates in cellular response to oxidative stress

[267] BALB/c and C57BL/6

mice

Up-regulation on induction of allergic airway disease. Antioxidant properties speculated as cause of up-regulation

[273]

Gsto1 knockout mice

(Gsto1-/-)

Reduction of Monomethylarsenate, Dimethylarsenic acid in the biotransformation of inorganic arsenic

[274, 275]

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1.10.2 GSTO1-1 and Glutathionylation

The crystal structure of GSTO1-1 is suggestive of a novel role of GSTO1-1 in glutathionylation, atypical of the superfamily. GSTO1-1 possesses the typical GST fold comprising of the N-terminal ‘thioredoxin’ resembling domain embedding the GSH binding site and the C-terminal helical domain [262]. As mentioned earlier, most GSTs possess a tyrosine or serine residue at their active site, responsible for catalysing conjugation reactions between GSH and electrophilic substrates. The active site residue in GSTO1-1 was identified as cysteine (Cys 32) flanked by proline and phenylalanine uncharacteristic of other mammalian GSTs [262]. On detailed dissection of the crystal structure, the active cysteine (Cys 32) was found to form a disulphide bond with GSH, a feature that is characteristic of glutaredoxin. Additionally, the GSH binding site is interestingly positioned in a wide crevice that can potentially accommodate large substrates such as proteins, suggesting potential interactions with molecules apart from GSH [197].

Due to the structural diversity at the substrate-binding site among the members of the GST superfamily and their ability to bind to GSH, it was hypothesized that the enzymes may be involved in protein-protein interactions leading to downstream effects. The enzymatic glutathionylation of proteins in vitro by GSTP1-1 was recently demonstrated by Townsend et al. using actin as an in vitro substrate [20]. However, it is yet to be determined whether other GSTs participate in the glutathionylation cycle.

This section attempts to summarize the literature on GSTO1-1 and justify the relevance of this information to the main objective of my project. There is increasing evidence for the physiological significance of GSTO1-1 in cellular defence mechanisms. Most GSTs are up-regulated by oxidative stress but the uniqueness of the structure of GSTO1-1 with respect to its active site suggests a novel role for this enzyme in physiological and oxidative stressed environments. The close structural resemblance of GSTO1-1 with Glutaredoxin, summarized in Table 1.3 supports the possibility that GSTO1-1 may catalyse glutaredoxin like activities.

Previous data from our laboratory confirm the thioltransferase activity of GSTO1-1 with chemical substrates such as 2-Hydroxyethyl disulphide (HEDS) [275]. Preliminary data from my project take this one step further and demonstrates the deglutathionylation of a glutathionylated cysteine residue in an 8-mer peptide and GSTO1-1 in vitro. This observation provides experimental evidence supporting a novel catalytic role of

40 GSTO1-1 in the deglutathionylation of proteins. In 2006, Sulfiredoxin was identified as a novel regulator of redox sensitive protein modification with the ability to deglutathionylate proteins [29]. Although the protein shares structural resemblance to the glutaredoxin family of enzymes, the catalytic cys-99 responsible for the thioltransferase activity is not conserved between the protein and Grx members. The cys-99 residue is instead in alignment with the non-conserved cysteine residue in Grx5 which is known to possess deglutathionylating activity. In the case of GSTO1-1, Cys 32 is conserved with the catalytic cysteine in Grx, a finding favouring the hypothesis underlying this study. Moreover, sulfiredoxin over-expression was demonstrated to regulate cell proliferation and drug response in vitro via the manipulation of proteins involved in regulating the cell cycle [25]. This study has encouraged me to question the possibility that GSTO1-1 may play a role in cell cycle modulation and drug resistance given that deglutathionylation is a common mechanism with the potential to influence global cell signalling.

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Structural/functional characteristics

Glutaredoxin GSTO1-1

Thioredoxin fold-like structure + + Active site cysteine residue Two catalytic cysteine

residues

One catalytic cysteine residue conserved with

glutaredoxin Glutathionylation activity Catalyses both

glutathionylation and deglutathionylation

To be characterized (Recently published study indicates demonstrates the glutathionylating activity

of GSTO1-1 in

Drosophila while data from our lab suggest deglutathionylation by GSTO1-1) Thioltransferase activity ++ + Dehydroascorbate reductase activity ++ +

Ability to accommodate large substrates such as proteins in the

active site

++ ++

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